DNA sequences of genes from fimbriae d&#39;escherichia coli strain DSM 6601

ABSTRACT

The invention relates to DNA sequences from fimbrial gene clusters of  Escherichia coli  strain DSM 6601. This strain has two chromosomal fimbrial gene clusters, namely type I (fim) and FIC (foc) gene clusters. DNA sequences of the main subunits fimA and focA are represented by SEQ ID NOS: 1 and 2.

The invention relates to DNA sequences from fimbrial gene clusters of Escherichia coli strain DSM 6601.

Escherichia coli is a gram-negative bacterium that occurs in human and animal intestinal flora as well as outside the intestines. E. coli exists in numerous varieties, which differ as regards capsule antigens, surface antigens and flagella antigens and can therefore be subdivided into numerous serological types. Classification by serotypes, however, does not provide any indication of the different virulence of the microorganisms. Representatives of one and the same serotype can have different pathogenic potential both in the human and in the animal body, ranging in the extreme case from avirulent to highly pathogenic. The E. coli strain DSM 6601 belongs to serogroup O6:K5, and is rated as nonpathogenic to humans or animals.

This strain has two chromosomally encoded fimbrial gene clusters, namely type I (fim) and F1C (foc) gene clusters. It is known that the fimbriae of this strain carry an adhesin. Adhesins are structures that perform an important function in the adhesion of the bacterial organism to other cells.

The main application of fimbrial genes is in analysis and diagnostics. Nevertheless, other possible applications exist in medicine and nutritional physiology and in biotechnology.

Fimbrial genes or their main subunits can be used, for example, to characterize a given strain, and so further studies of the sequence of these genes is needed.

According to the invention, studies have now been performed with E. coli strain DSM 6601, and the obtained DNA sequences of the main subunits fimA (FIG. 1) (SEQ ID NO:1) and focA (FIG. 2) (SEQ ID NO:2) of the fimbriae have been precisely analyzed. The DNA sequences obtained from the strain were subjected to DNA sequence analysis by means of database programs and were compared with the DNA sequences of known strains. Whereas differences compared with the strain AD 110 (SEQ ID NO:3) were found at one location of the DNA sequence in the main subunits focA of strain DSM 6601, differences relative to the comparison strain HB 101 (SEQ ID NO:4) were found at several locations in the DNA sequence of the fimA gene of strain DSM 6601, as FIGS. 1 and 2 show:

For analysis of the two main subunits fimA and focA of the fimbriae of strain DSM 6601—and of the comparison strains AD 110 and HB 101—the corresponding DNA fragments were first amplified from the chromosome of the strains by means of PCR reactions.

The invention will now be explained in more detail by means of examples:

EXAMPLES 1

Amplification of the fimA Gene From Strains DSM 6601 and HB 101

The following primer pairs were used for this PCR reaction:

fimA1: 5′-GTG TAC AGA ACG ACT GCC-3′  (SEQ ID NO:5)

fimA2: 5′-GTA ATG ACG TCC CTG AAC-3′  (SEQ ID NO:6)

PCR conditions:

step 1: denature for 3 min at 95° C.

step 2: 45 sec at 95° C.

step 3: 45 sec at 53° C.

step 4: 45 sec at 72° C.

step 5: 5 min at 72° C.

Steps 2 to 4 were repeated 30 times.

EXAMPLES 2

Amplification of the focA Gene From Strains DSM 6601 and AD 110

The following primer pairs were used for this PCR reaction:

focA1: 5′-CTC ACA TTG CAT TTA TGA AG-3′  (SEQ ID NO:7)

focA2: 5′-GCT ATA TAT CCG TTA CAC TG-3′  (SEQ ID NO:8)

PCR conditions:

step 1: denature for 3 min at 95° C.

step 2: 45 sec at 95° C.

step 3: 45 sec at 51° C.

step 4: 45 sec at 72° C.

step 5: 5 min at 72° C.

Steps 2 to 4 were repeated 30 times.

EXAMPLE 3″

Cloning and Plasmid Isolation

The PCR products obtained in Examples 1 and 2 were cloned in vector pUC 18 by the procedure of Sambrook et al. (Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory, second edition (1989), Cloning, Transformation: 1.53-1.84; PCR: 14.00-14.35), and the resulting plasmid DNA was transformed into the E. coli K12 strain DH5α.

The plasmid DNA was isolated by the procedure or Birnboim et al. (Birnboim, A. C. and Doly, J. (1979) Nucl. Acids Res. 7:1513-1523. A rapid alkaline extraction procedure for screening recombinant plasmid DNA).

3 ml of LB medium was inoculated with a bacteria colony and shaken overnight at 37 ° C. This culture was centrifuged in an Eppendorf tube, and the residue of medium was removed with a pipette. The cell sediment was resuspended with 100 μl of solution I (50 mM glucose; 10 mM EDTA, pH 8; 25 mM Tris-HCl, pH 8). After 5 minutes of incubation at room temperature, there was added thereto 200 μl of solution II (0.2 N NaOH; 1% SDS) and mixing was continued until the contents became clear, after which the Eppendorf tube was allowed to stand for a further 5 minutes on ice. Thereafter there was added 150 μl of solution III (3 M Na acetate, pH 4.8), shaking was performed briefly until precipitation of the chromosomal DNA in flocculent form, and the mixture was left on ice for another 5 minutes. The precipitated chromosomal DNA and the cell residues were pelleted for 5 minutes in the centrifuge, and the supernatant containing the plasmid DNA was transferred into a new tube. For purification of the plasmid DNA there were added 50 μl of phenol and 150 μl of chloroform/isoamyl alcohol (24:1), after which the contents were shaken briefly and centrifuged for 2 minutes. The aqueous phase was pipetted into a new tube. The plasmid DNA was precipitated with 2 volumes of ice-cold ethanol and centrifuged for 10 minutes. The pellet was washed with 70% ethanol and dried in the Speedvac. The plasmid DNA was resuspended in 20 μl of doubly distilled water and stored at −20° C.

EXAMPLE 4

DNA Sequencing

DNA sequencing was performed by the procedure of Sanger et al. (Sanger, F., Nicklen, S. and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467. DNA sequencing with chain-terminating inhibitors).

DNA sequencing was performed with the T7 sequencing kit of the Pharmacia-LKB Co.

For the denaturing step, 8 μl (1.5 to 2 μg) of plasmid DNA was mixed with 2 μl of 2 N NaOH, briefly centrifuged and incubated for 10 minutes at room temperature. The DNA was precipitated for 15 minutes at −70° C. with 3 μl of 3 M Na acetate, pH 4.8 as well as 7 μl of doubly distilled water and 60 μl of ice-cold absolute ethanol. The precipitated DNA was centrifuged for 10 minutes, washed with 70% ethanol and dried.

For the annealing reaction, the denatured DNA was suspended in 10 μl of doubly distilled water and mixed with 2 μl of annealing buffer and 2 μl (40 ng) of primer. The mixture was incubated for 20 minutes at 37° C., to allow the primer to bind to the template DNA. The reaction mixture was cooled for 10 minutes at room temperature and then either used immediately for the labeling reaction or frozen at −20° C. For the labeling reaction, 3 μl of labeling mix, 1 μl of [α-³²P]dATP and 2 μl of T7 polymerase (diluted in 1:5 ratio with enzyme dilution buffer) were pipetted into the annealing reaction mixture and, after brief mixing, were incubated for 5 minutes at room temperature. Meanwhile the sequencing mixes (2.5 μl each of ‘G’, ‘A’, ‘T’ and ‘C’ mix “short” per tube) already prepared for the termination reaction were preheated to 37° C. After completion of the labeling reaction, 4 μl aliquots thereof were added to each of the four sequencing mixes and mixed briefly with the pipette. The termination reactions were incubated for 5 minutes at 37° C. To end the termination reactions, 5 μl of stop solution were added in each case. The mixtures were then transferred into an incubator at 95° C., denatured for 2 minutes and then placed on ice. 2.5 μl aliquots of the reactions were placed on a sequencing gel [25.2 g of urea, 22 ml of doubly distilled water, 6 ml of 10×TBE, 10 ml of polyacrylamide (40%), 2 ml of ammonium persulfate (16 mg/ml) and 60 μl of TEMED), in the succession ‘G’, ‘A’, ‘T’, ‘C’. The electrophoresis was performed at 40 watt and 1500 volt for 4.5 hours.

These DNA sequences can also be prepared synthetically in ways known in themselves, and particular sequence segments can be used as primer, in which case identification of E. coli strain DSM 6601 then becomes possible without difficulty. Of course, the possibility also exists that the corresponding DNA sequences of this strain can be introduced by genetic engineering into other E. coli strains, in order, for example, to modify the behavior as regards adhesion of the cells and thus to influence the colonization properties of other strains.

SEQUENCE PROTOCOL GENERAL INFORMATION APPLICANT: PHARMA-ZENTRALE GMBH LOERFELDSTRASSE 20 58313 HERDECKE FEDERAL REPUBLIC OF GERMANY TITLE OF THE INVENTION: DNA SEQUENCES FROM FIMBRIAL GENES OF ESCHERICHIA COLI STRAIN DSM 6601 NUMBER OF SEQUENCES: 2 COMPUTER-READABLE VERSION: DATA MEDIUM DISKETTE COMPUTER IBM COMPATIBLE OPERATING SYSTEM WINDOWS 95 SOFTWARE MICROSOFT WORD 6.0 DATA OF THE PRESENT APPLICATION APPLICATION NUMBER APPLICATION DATE AGENT INFORMATION NAME DRES. HARMSEN & UTESCHER AGENT NUMBER 268569 FILE NUMBER PT 19/97 TEL 040-249757 FAX 040-2803672 SEQUENCE ID NO. INFORMATION: fimadsm SEQUENCE CHARACTERISTICS: LENGTH 549 BASE PAIRS TYPE DNA STRAND FORM DOUBLE STRAND TOPOLOGY LINEAR ORIGINAL SOURCE: ORGANISM ESCHERICHIA COLI STRAIN DSM 6601 CELL TYPE SINGLE-CELLED ORGANISM BIBLIOGRAPHY: AUTHORS KLEMM, P. TITLE The fimA gene encoding the type 1 fimbrial subunit of Escherichia coli PERIODICAL EUR. J., BIOCHEM. VOLUME 143 (2) PAGES 395-399 DATE 1984

SEQUENCE DESCRIPTION SEQ ID NO: fimadsm 1 ATGAAAATTA AAACTCTGGC AATCGTTGCT CTGTCGGCTC TGTCCCTCAG 51 TTCCGCAGCG GCTCTGGCCG ATACTACGAC GGTAAATGGT GGGGCCGTTC 101 ACTTTAAAGG GGAAGTTGTT AACGCCGCTT GCGCAGTTGA TGCAGGCTCT 151 GTTGATCAAA CCGTTCAGTT AGGCCAGGTT CGTACCGCTA GCCTGAAGCA 201 GGAAGGAGCA ACCAGCTCTG CCGTTGGTTT TAACATTCAG GTGAATGATT 251 GCGATACCAC TGTTGCCACA AAAGCTGCTG TTGCCTTCTT AGGTACGGCA 301 ATTGATGCTA CCGATACTGA TGTACTGGCT CTGCAGAGTT CAGCTGCGGG 351 TAGCGCAACA AACGTTGGTG TGCAGATCCT GGACAGAACG GGTGCTGCGC 401 TGACGCTGGA CGGTGCGACA TTTAGTTCAG AAACAACCCT GAATAACGGA 451 ACCAATACCA TTCCGTTCCA GGCGCGTTAT TTTGCAACCG GTGCCGCAAC 501 CCCGGGTGCT GCTAATGCGG ATGCGACCTT CAAGGTTCAG TATCAATAA

SEQUENCE PROTOCOL GENERAL INFORMATION APPLICANT: PHARMA-ZENTRALE GMBH LOERFELDSTRASSE 20 58313 HERDECKE FEDERAL REPUBLIC OF GERMANY TITLE OF THE INVENTION: DNA SEQUENCES FROM FIMBRIAL GENES OF ESCHERICHIA COLI STRAIN DSM 6601 NUMBER OF SEQUENCES: 2 COMPUTER-READABLE VERSION: DATA MEDIUM DISKETTE COMPUTER IBM COMPATIBLE OPERATING SYSTEM WINDOWS 95 SOFTWARE MICROSOFT WORD 6.0 DATA OF THE PRESENT APPLICATION APPLICATION NUMBER APPLICATION DATE AGENT INFORMATION NAME DRES. HARMSEN & UTESCHER AGENT NUMBER 268569 FILE NUMBER PT 19/97 TEL 040-249757 FAX 040-2803672 SEQUENCE ID NO. INFORMATION: focadsm SEQUENCE CHARACTERISTICS: LENGTH 543 BASE PAIRS TYPE DNA STRAND FORM DOUBLE STRAND TOPOLOGY LINEAR ORIGINAL SOURCE: ORGANISM ESCHERICHIA COLI STRAIN DSM 6601 CELL TYPE SINGLE-CELLED ORGANISM SEQUENCE DESCRIPTION SEQ ID NO: focadsm

SEQUENCE DESCRIPTION SEQ ID NO: focadsm 1 atgaagttaa aatccatctc catggctgta ttttcagctc tgtccctggg 51 tgttgcgaca aatgcgtctg ctgtcaccac ggttaggtgt ggtacagttc 101 attttaaggg tgaagtggtt aatgctgcat gtgctgtaaa cactaactca 151 ttcgatcaga cggttaatct tggacaggtt cgttccgaaa gattgaaagt 201 agatggagct aaaagcaatc cagttggatt tacaattgaa ttaaatgatt 251 gtgactcgca ggtgtctgct ggtgcaggaa ttgtcttttc aggcccagca 301 gttactggta aaacagatgt tcttgcttta caaagttctg cagcgggttc 351 tgcaacaaac ttcggcgttc agattactga ccataggccg aaggttgtac 401 ctttagatgg aactgcaagc tcaacgttta cattaactga cggaaccaac 451 aaaattccat ttcaggcggt ttactacgca actggacagg ccactgctgg 501 tattgccaac gccgacgcca cctttaaagt tcagtaccag taa

8 1 549 DNA Escherichia coli 1 atgaaaatta aaactctggc aatcgttgct ctgtcggctc tgtccctcag ttccgcagcg 60 gctctggccg atactacgac ggtaaatggt ggggccgttc actttaaagg ggaagttgtt 120 aacgccgctt gcgcagttga tgcaggctct gttgatcaaa ccgttcagtt aggccaggtt 180 cgtaccgcta gcctgaagca ggaaggagca accagctctg ccgttggttt taacattcag 240 gtgaatgatt gcgataccac tgttgccaca aaagctgctg ttgccttctt aggtacggca 300 attgatgcta ccgatactga tgtactggct ctgcagagtt cagctgcggg tagcgcaaca 360 aacgttggtg tgcagatcct ggacagaacg ggtgctgcgc tgacgctgga cggtgcgaca 420 tttagttcag aaacaaccct gaataacgga accaatacca ttccgttcca ggcgcgttat 480 tttgcaaccg gtgccgcaac cccgggtgct gctaatgcgg atgcgacctt caaggttcag 540 tatcaataa 549 2 543 DNA Escherichia coli 2 atgaagttaa aattcatctc catggctgta ttttcagctc tgaccctggg tgttgcgaca 60 aatgcgtctg ctgtcaccac ggttaggtgt ggtacagttc attttaaggg tgaagtggtt 120 aatgctgcat gtgctgtaaa cactaactca ttcgatcaga cggttaatct tggacaggtt 180 cgttccgaaa gattgaaagt agatggagct aaaagcaatc cagttggatt tacaattgaa 240 ttaaatgatt gtgactcgca ggtgtctgct ggtgcaggaa ttgtcttttc aggcccagca 300 gttactggta aaacagatgt tcttgcttta caaagttctg cagcgggttc tgcaacaaac 360 ttcggcgttc agattactga ccataggccg aaggttgtac ctttagatgg aactgcaagc 420 tcaacgttta cattaactga cggaaccaac aaaattccat ttcaggcggt ttactacgca 480 actggacagg ccactgctgg tattgccaac gccgacgcca cctttaaagt tcagtaccag 540 taa 543 3 543 DNA Escherichia coli 3 atgaagttaa aattcatctc catggctgta ttttcagctc tgaccctggg tgttgcgaca 60 aatgcgtctg ctgtcaccac ggttaatggt ggtacagttc attttaaggg tgaagtggtt 120 aatgctgcat gtgctgtaaa cactaactca ttcgatcaga cggttaatct tggacaggtt 180 cgttccgaaa gattgaaagt agatggagct aaaagcaatc cagttggatt tacaattgaa 240 ttaaatgatt gtgactcgca ggtgtctgct ggtgcaggaa ttgtcttttc aggcccagca 300 gttactggta aaacagatgt tcttgcttta caaagttctg cagcgggttc tgcaacaaac 360 ttcggcgttc agattactga ccataggccg aaggttgtac ctttagatgg aactgcaagc 420 tcaacgttta cattaactga cggaaccaac aaaattccat ttcaggcggt ttactacgca 480 actggacagg ccactgctgg tattgccaac gccgacgcca cctttaaagt tcagtaccag 540 taa 543 4 597 DNA Escherichia coli 4 gactgcccat gtcgatttag aaatagtttt ttgaaaggaa agcagcatga aaattaaaac 60 tctggcaatc gttgttctgt cggctctgtc cctcagttct acagcggctc tggccgctgc 120 cacgacggtt aatggtggga ccgttcactt taaaggggaa gttgttaacg ccgcttgcgc 180 agttgatgca ggctctgttg atcaaaccgt tcagttagga caggttcgta ccgcatcgct 240 ggcacaggaa ggagcaacca gttctgctgt cggttttaac attcagctga atgattgcga 300 taccaatgtt gcatctaaag ccgctgttgc ctttttaggt acggcgattg atgcgggtca 360 taccaacgtt ctggctctgc agagttcagc tgcgggtagc gcaacaaacg ttggtgtgca 420 gatcctggac agaacgggtg ctgcgctgac gctggatggt gcgacattta gttcagaaac 480 aaccctgaat aacggaacca ataccattcc gttccaggcg cgttattttg ccggggccgc 540 aaccccgggt gctgctaatg cggatgcgac cttcaaggtt cagtatcaat aacctac 597 5 18 DNA Artificial Sequence Description of Artificial Sequence Primer 5 gtgtacagaa cgactgcc 18 6 18 DNA Artificial Sequence Description of Artificial Sequence Primer 6 gtaatgacgt ccctgaac 18 7 20 DNA Artificial Sequence Description of Artificial Sequence Primer 7 ctcacattgc atttatgaag 20 8 20 DNA Artificial Sequence Description of Artificial Sequence Primer 8 gctatatatc cgttacactg 20 

What is claimed is:
 1. A nucleic acid having the nucleotide sequence designated SEQ. ID NO.
 1. 2. A reagent comprising a nucleic acid of claim
 1. 3. A nucleic acid amplification kit comprising a nucleic acid of claim
 1. 4. A recombinant Escherichia coli containing a nucleic acid of claim
 1. 5. A nucleic acid having the nucleotide sequence designated SEQ. ID NO.
 2. 6. A reagent comprising a nucleic acid of claim
 5. 7. A nucleic acid amplification kit comprising a nucleic acid of claim
 5. 8. A recombinant Escherichia coli containing a nucleic acid of claim
 5. 